U.S. patent application number 16/785599 was filed with the patent office on 2020-10-22 for bio-refinery waste utilization for enzyme production using novel penicillium funiculosum mrj-16 fungal strain.
This patent application is currently assigned to INDIAN OIL CORPORATION LIMITED. The applicant listed for this patent is DEPARTMENT OF BIOTECHNOLOGY, INDIAN OIL CORPORATION LIMITED. Invention is credited to Mukund ADSUL, Ravi Prakash GUPTA, Anshu Shankar MATHUR, Suresh Kumar PURI, Sankara Sri Venkata RAMAKUMAR, Simranjeet Kaur SANDHU, Reeta Rani SINGHANIA, Deepak Kumar TULI.
Application Number | 20200332329 16/785599 |
Document ID | / |
Family ID | 1000004686179 |
Filed Date | 2020-10-22 |
United States Patent
Application |
20200332329 |
Kind Code |
A1 |
ADSUL; Mukund ; et
al. |
October 22, 2020 |
BIO-REFINERY WASTE UTILIZATION FOR ENZYME PRODUCTION USING NOVEL
PENICILLIUM FUNICULOSUM MRJ-16 FUNGAL STRAIN
Abstract
The present invention relates to a method for obtaining a high
titer of enzyme mixture comprising cellulases, hemicellulases and
.beta.-glucosidases in reutilization of waste water generated
during hot water extraction of lignocellulosic biomass or
biorefinery waste water using Penicillium funiculosum MRJ-16 mutant
strain. The cellulose or lignocellulosic biomass used in the
fermentation process is selected from the group consisting of rice
straw, wheat straw, corn stover, cotton stalk or a combination
thereof. The enzyme mixture obtained by the present process is used
for the saccharification of acid pretreated lignocellulosic
biomass.
Inventors: |
ADSUL; Mukund; (Faridabad,
IN) ; SANDHU; Simranjeet Kaur; (Faridabad, IN)
; SINGHANIA; Reeta Rani; (Faridabad, IN) ; GUPTA;
Ravi Prakash; (Faridabad, IN) ; PURI; Suresh
Kumar; (Faridabad, IN) ; RAMAKUMAR; Sankara Sri
Venkata; (Faridabad, IN) ; MATHUR; Anshu Shankar;
(Faridabad, IN) ; TULI; Deepak Kumar; (Faridabad,
IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INDIAN OIL CORPORATION LIMITED
DEPARTMENT OF BIOTECHNOLOGY |
Mumbai
New Delhi |
|
IN
IN |
|
|
Assignee: |
INDIAN OIL CORPORATION
LIMITED
Mumbai, Maharashtra
IN
DEPARTMENT OF BIOTECHNOLOGY
New Delhi
IN
|
Family ID: |
1000004686179 |
Appl. No.: |
16/785599 |
Filed: |
February 8, 2020 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 9/2437 20130101;
C12R 1/80 20130101; C12P 19/14 20130101 |
International
Class: |
C12P 19/14 20060101
C12P019/14; C12N 9/42 20060101 C12N009/42 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 17, 2019 |
IN |
201921015449 |
Claims
1. A method for obtaining a high titer of enzyme mixture comprising
cellulases, hemicellulases and .beta.-glucosidases in reutilization
of waste water, the process comprising the steps of: (i) preparing
fermentation media comprising ammonium sulphate (3-5 g/L),
KH.sub.2PO.sub.4 (4-6 g/L), MgSO.sub.4.7H.sub.2O (0.5-1 g/L),
CaCO.sub.3 (2.5-5 g/L), Glycerol (2-3 g/L), Corn steep solids
(25-35 g/L), carbon source (25-40 g/L), Tween-80 (1-2 ml/L) and
waste water obtained during pre-treatment of lignocellulosic
biomass or biorefinery waste water; (ii) inoculating the media
components of step (a) with 10% active liquid seed culture of
Penicillium funiculosum MRJ-16; (iii) subjecting the Penicillium
funiculosum MRJ-16 culture of step (b) to fermentation in an
aerated fermenter; (iv) harvesting the enzyme broth after the
fermentation process of step (c) and subjecting the broth to
centrifugation to obtain the enzyme mixture.
2. The method as claimed in claim 1, wherein waste water comprises
of waxes, proteins, phenolics and inhibitors.
3. The method as claimed in claim 2, wherein inhibitors in the
waste water are organic acid, phenol, hydroxyl methyl furfural
(HMF), formic acid, acetic acid and furfural.
4. The method as claimed in claim 1, wherein the carbon source in
the fermentation media of step (a) is selected from the group
consisting of cellulose, acid pretreated lignocellulosic biomass or
a combination thereof.
5. The method as claimed in claim 1, wherein the fermentation
process is carried out at a temperature in the range of
25-35.degree. C., pH in the range of 4-6, aeration in the range of
0.5-2.5VVM and dissolved oxygen above 30%.
6. The method as claimed in claim 1, wherein the fermentation is
carried out for a time period of 80-110 hours.
7. The method as claimed in claim 4, wherein cellulose or
lignocellulosic biomass is selected from the group consisting of
rice straw, wheat straw, corn stover, cotton stalk or a combination
thereof.
8. The method as claimed in claim 1, wherein the enzyme mixture
obtained is used for the saccharification of acid pretreated
lignocellulosic biomass.
Description
FIELD OF THE INVENTION
[0001] Present invention relates to a method for obtaining a high
titer of enzyme mixture in reutilization of waste water generated
during pretreatment of lignocellulosic biomass. More specifically,
the present invention relates to a method for obtaining high titer
of enzyme mixture comprising cellulases, hemicellulases and
.beta.-glucosidases in reutilization of waste water generated
during pretreatment of lignocellulosic biomass using Penicillium
funiculosum MRJ-16 mutant strain.
BACKGROUND OF THE INVENTION
[0002] Lignocellulosic biomass comprises of three main fractions;
cellulose, hemicelluloses and lignins, varying in their composition
and arrangement depending upon the crop type and environmental
conditions. The process of transforming lignocellulosic biomass to
ethanol generally comprises three steps: first pretreatment (acid,
alkali or steam explosion) that make cellulose and hemicelluloses
accessible to enzymes, second enzymatic hydrolysis of cellulose and
hemicelluloses to release hexose and pentose sugars and third is
utilization of pentose and hexose sugars by fermenting yeast to
produce ethanol.
[0003] Pretreatment of lignocellulosic biomass can involve multiple
steps like washing, soaking, chemical, high temperature, steam
explosion etc. Most commonly used acid pretreatment of
lignocellulosic biomass aimed to increase the surface area of
cellulose by hydrolyzing major fraction of hemicelluloses and small
fraction of cellulose so that enzymes easily access the cellulose
and release glucose. Acid pretreatment hydrolyze the hemicellulose
fraction to release xylose, glucose, mannose and arabinose and
depending upon the severity of treatment these sugars get converted
into toxic substances such as hydroxyl methyl furfurals, formic
acid, acetic acid and furfurals. Presence of these inhibitors
negatively affects the growth of micro-organisms and enzyme
action.
[0004] Enzymatic breakdown of cellulose and hemicelluloses exposed
required various cellulases, hemicellulases and .beta.-glucosidase
enzymes. These enzymes are produced industrially by fungi belongs
to genera Trichoderma, Aspergillus and Humicola sp. by submerged
fermentation in either continuous or batch mode. On commercial
scale fungal biomass is generally separated from the enzyme broth
and clear enzyme broth is concentrated, formulated, stabilized and
stored further use. For industrial purposes these fungal strains
are mutagenize to improve its properties like high enzyme yield,
high specific activity, minimum chemical requirement and
versatility of enzymes.
[0005] US 2011/0262997 disclose a process of cellulases enzyme
production by Trichoderma reesei in an economical way by using
pre-treated lignocellulosic material as inducer. Fermentation was
carried out for 165 h at 28.degree. C. in Fed-Batch mode using
soluble glucose as feed. Combined physical and chemical
pre-treatment was given to corn stover. Pre-treatment was carried
out at a pressure of about 450 psi at 235.degree. C. using sulfuric
acid (0.5-1.4%). However, the pre-treated material used was
detoxified by washing, repeated soaking in water to remove
compounds that inhibit the performance of microorganism.
Detoxification method is time consuming, lot of water wastage and
another additional cost factor to the overall process.
[0006] US 2016/9249402 disclose the production of cellulases and
hemicellulases enzyme in two stages in which growth of fungus
Trichoderma reesei CL847 occurs in batch mode for 50 h followed by
continuous mode of enzyme production phase for more than 300 h in
aerated stirred tank bioreactor. During enzyme production phase,
inducer (acid pre-treated lignocellulosic hydrolysate containing
soluble sugars) was added continuously at 8 ml/h of concentration.
Acid pre-treated lignocellulosic hydrolysate was obtained from
straw pre-treated by steam explosion with prior impregnation of
sulfuric acid. The inducer solution used was not sterilized and no
pH adjustment was done. The pH of fermentation media was controlled
during entire process using 5.5M ammoniacal solution. The mass of
the reaction was kept constant. The final productivity was 0.39
g/L/h with FPU of 77.6 IU/ml and .beta.-glucosidase of 1.3 IU/mg.
Although this process eliminates the need of hydrolysate pH
rectification step and final productivity was high but
.beta.-glucosidase activity was low, which means the enzyme
obtained will be incapable to efficiently hydrolyze the
lignocellulosic biomass and extra .beta.-glucosidase enzyme need to
be added and thus the second generation ethanol production process
lack competitiveness.
[0007] US 2014/045227 teach a process of efficient hydrolysis of
lignocelluloses biomass and process integrated with enzyme
production. Wheat straw was pretreated with sulphuric acid by
hydrothermal treatment under pressure. Biomass slurry obtained was
divided into two parts: (a) one part was used for enzyme production
with 8% w/v pretreated biomass and 2% corn steep liquor in media.
Fermentation was carried out by Trichoderma reesei for 5 days. (b)
Other part of pretreated wheat straw slurry was hydrolysed by
enzyme prepared above along with mechanically sheared fungal
mycelia and unutilized biomass. Enzyme dosage used was 0.5% w/w
(protein/pre-treated straw) along with .beta.-glucoside enzyme from
Novozyme was added at 2 cellobiose units/mg cellulases at 20%
substrate loading. Maximum of 40 g/L of glucose yield was obtained
after 72 hrs of hydrolysis. In this process, no details of enzyme
activities were given, additional .beta.-glucoside enzyme was added
during hydrolysis and enzyme dosage is quite high, all these points
makes this process unfeasible at industrial scale.
[0008] In comparison to the prior art, present invention is aimed
at providing method of cellulases enzyme production at high titer
using economical media components by novel Penicillium funiculosum
MRJ-16 mutant strain that is catabolite derepressed and can
tolerate high concentration of inhibitors. The novel Penicillium
funiculosum MRJ-16 mutant strain is having 6.47 FPU/ml and 62 BGL
(IU/ml) whereas Penicillium funiculosum NCIM 1228 (Parent strain)
is having 3.5 FPU/ml and 22.1 BGL (IU/ml) activity hence, the novel
Penicillium funiculosum MRJ-16 mutant strain possess enhanced
enzyme activity as compared to cited arts. Present invention will
help to bring down the production cost of bioethanol and make the
production sustainable and competitive along with processing waste
effluents generated during pretreatment.
SUMMARY OF THE INVENTION
[0009] The present provides a method for obtaining a high titer of
enzyme mixture comprising cellulases, hemicellulases and
.beta.-glucosidases in reutilization of waste water, the process
comprising the steps of: [0010] (i) preparing fermentation media
comprising ammonium sulphate (3-5 g/L), KH.sub.2PO.sub.4 (4-6 g/L),
MgSO.sub.4.7H.sub.2O (0.5-1 g/L), CaCO.sub.3 (2.5-5 g/L), Glycerol
(2-3 g/L), Corn steep solids (25-35 g/L), carbon source (25-40
g/L), Tween-80 (1-2 ml/L) and waste water obtained during
pre-treatment of lignocellulosic biomass or biorefinery waste
water; [0011] (ii) inoculating the media components of step (a)
with 10% active liquid seed culture of Penicillium funiculosum
MRJ-16; [0012] (iii) subjecting the Penicillium funiculosum MRJ-16
culture of step (b) to fermentation in an aerated fermenter; [0013]
(iv) harvesting the enzyme broth after the fermentation process of
step (c) and subjecting the broth to centrifugation to obtain the
enzyme mixture.
[0014] In an embodiment of the present invention, the waste water
used in step (i) contains waxes, proteins, phenolics and
inhibitors.
[0015] In an embodiment of the present invention, inhibitors in the
waste water are organic acid, phenol, hydroxyl methyl furfural
(HMF), formic acid, acetic acid and furfural.
[0016] In an embodiment of the present invention, the carbon source
in the fermentation media of step (a) is selected from the group
consisting of cellulose, acid pretreated lignocellulosic biomass or
a combination thereof.
[0017] In an embodiment of the present invention, the fermentation
process is carried out at a temperature in the range of
25-35.degree. C., pH in the range of 4-6, aeration in the range of
0.5-2.5VVM and dissolved oxygen above 30%.
[0018] In an embodiment of the present invention, the fermentation
process is carried out for a time period of 80-110 hours.
[0019] In an embodiment of the present invention, cellulose or
lignocellulosic biomass used in the fermentation process is
selected from the group consisting of rice straw, wheat straw, corn
stover, cotton stalk or a combination thereof.
[0020] In an embodiment of the present invention, the enzyme
mixture obtained is used for the saccharification of acid
pretreated lignocellulosic biomass.
DETAILED DESCRIPTION OF THE INVENTION
[0021] While the invention is susceptible to various modifications
and/or alternative processes and/or solvent system, specific
embodiment thereof has been shown by way of examples and will be
described in detail below. It should be understood, however that it
is not intended to limit the invention to the particular processes
and/or temperature, pH, ratios, quantity and strains disclosed, but
on the contrary, the invention is to cover all modifications,
equivalents, and alternative falling within the spirit and the
scope of the invention as defined by the appended claims.
[0022] The following description is of exemplary embodiments only
and is not intended to limit the scope, applicability or
configuration of the invention in any way. Rather, the following
description provides a convenient illustration for implementing
exemplary embodiments of the invention. Various changes to the
described embodiments may be made in the function and arrangement
of the elements described without departing from the scope of the
invention.
[0023] Any particulars and all details set forth herein are used in
the context of some embodiments and therefore should not be
necessarily taken as limiting factors to the attached claims. The
attached claims and their legal equivalents can be realized in the
context of embodiments other than the ones used as illustrative
examples in the description below.
[0024] For convenience, before further description of the present
disclosure, certain terms employed in the specification, and
examples are collected here. These definitions should be read in
the light of the remainder of the disclosure and understood as by a
person of skill in the art. The terms used herein have the meanings
recognized and known to those of skill in the art, however, for
convenience and completeness, particular terms and their meanings
are set forth below.
[0025] The articles "a", "an" and "the" are used to refer to one or
to more than one (i.e., to at least one) of the grammatical object
of the article.
[0026] The terms "comprise" and "comprising" are used in the
inclusive, open sense, meaning that additional elements may be
included. It is not intended to be construed as "consists of
only".
[0027] Throughout this specification, unless the context requires
otherwise the word "comprise", and variations such as "comprises"
and "comprising", will be understood to imply the inclusion of a
stated element or step or group of element or steps but not the
exclusion of any other element or step or group of element or
steps.
[0028] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosure belongs.
Although any methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
disclosure, the preferred methods, and materials are now
described.
[0029] The present invention provides a method for obtaining a high
titer of enzyme mixture comprising cellulases, hemicellulases and
.beta.-glucosidases in reutilization of waste water by Penicillium
funiculosum MRJ-16 (MTCC Accession No. 25142 and date of deposition
is 12 Jun. 2017) using waste water generated during pretreatment of
lignocellulosic biomass. The present invention also provides a
method for the production of high titer of cellulases and
hemicellulases enzymes using Penicillium funiculosum MRJ-16 using
waste water generated during pretreatment of lignocellulosic
biomass.
[0030] The present provides a method for obtaining a high titer of
enzyme mixture comprising cellulases, hemicellulases and
.beta.-glucosidases in reutilization of waste water, the process
comprising the steps of: [0031] (i) preparing fermentation media
comprising ammonium sulphate (3-5 g/L), KH.sub.2PO.sub.4 (4-6 g/L),
MgSO.sub.4.7H.sub.2O (0.5-1 g/L), CaCO.sub.3 (2.5-5 g/L), Glycerol
(2-3 g/L), Corn steep solids (25-35 g/L), carbon source (25-40
g/L), Tween-80 (1-2 ml/L) and waste water obtained during
pre-treatment of lignocellulosic biomass or biorefinery waste
water; [0032] (ii) inoculating the media components of step (a)
with 10% active liquid seed culture of Penicillium funiculosum
MRJ-16; [0033] (iii) subjecting the Penicillium funiculosum MRJ-16
culture of step (b) to fermentation in an aerated fermenter; [0034]
(iv) harvesting the enzyme broth after the fermentation process of
step (c) and subjecting the broth to centrifugation to obtain the
enzyme mixture.
[0035] Waste water generated by hot water extraction of
lignocellulosic biomass contains waxes, proteins and phenolics.
Inhibitors are generated during acid pretreatment of
lignocellulosic biomass. Inhibitors are organic acids and phenols
such as hydroxyl methyl furfural (HMF), formic acid, acetic acid
and furfural.
[0036] In an embodiment, the present invention provides a method
for the production of high titer of cellulases and hemicellulases
enzymes using low cost minimum media components such as waste
effluents generated during pretreatment of lignocellulosic biomass
or biorefinery wastewater along with hyper-cellulolytic variants of
Penicillium funiculosum MRJ-16.
[0037] In an embodiment of the present invention, the waste water
used in step (i) contains waxes, proteins, phenolics and
inhibitors.
[0038] In an embodiment of the present invention, inhibitors in the
waste water are organic acid, phenol, hydroxyl methyl furfural
(HMF), formic acid, acetic acid and furfural.
[0039] In an embodiment of the present invention, waste water used
in step (i) of the process containing up to 0.1% concentration of
inhibitors is used for enzyme production.
[0040] In an embodiment of the present invention, the carbon source
in the fermentation media of step (a) is selected from the group
consisting of cellulose, acid pretreated lignocellulosic biomass or
a combination thereof.
[0041] In an embodiment of the present invention, the fermentation
process is carried out at a temperature in the range of
25-35.degree. C., pH in the range of 4-6, aeration in the range of
0.5-2.5VVM and dissolved oxygen above 30%.
[0042] In an embodiment of the present invention, the fermentation
process is carried out for a time period of 80-110 hours.
[0043] In an embodiment of the present invention, cellulose or
lignocellulosic biomass used in the fermentation process is
selected from the group consisting of rice straw, wheat straw, corn
stover, cotton stalk or a combination thereof.
[0044] In an embodiment of the present invention, the enzyme
mixture obtained is used for the saccharification of acid
pretreated lignocellulosic biomass.
[0045] Following non-limiting examples are given by way of
illustration for specific embodiments thereof and therefore should
not be construed to limit the scope of the invention.
EXAMPLES
[0046] The following examples are set forth below to illustrate the
methods and results according to the disclosed subject matter.
These examples are not intended to be inclusive of all aspects of
the subject matter disclosed herein, but rather to illustrate
representative methods, compositions, and results. These examples
are not intended to exclude equivalents and variations of the
present invention, which are apparent to one skilled in the
art.
Example 1--Production of Enzymes Using Preparatory Media
[0047] Fermentation process was carried out using ammonium sulphate
(3-5 g/L), KH.sub.2PO.sub.4 (4-6 g/L), MgSO.sub.4.7H.sub.2O (0.5-1
g/L), CaCO.sub.3 (2.5-5 g/L), Glycerol (2-3 g/L), Corn steep solids
(25-35 g/L), cellulose (25-40 g/L) and Tween-80 (1-2 ml/L)
dissolved in distilled water. Novel Penicillium funiculosum MRJ-16
mutant strain developed in the laboratory was used for the study.
Sterilized media was inoculation with 10% active liquid seed
culture of Penicillium mutant strain. After 96 hours of
fermentation, the enzyme broth was harvested and clear enzyme broth
was analyzed. The results obtained were 16.5 g/L of protein, 78
IU/ml of .beta.-glucosidase and 7.2 FPU/ml of filter paper
activity.
Example 2--Effect of Inhibitors on Enzyme Production
[0048] Cultivation of Penicillium funiculosum MRJ-16 mutant strain
was conducted under conditions and media composition identical to
example no. 1 along with inhibitors at different concentration.
Inhibitors such as hydroxyl methyl furfural (HMF), formic acid,
acetic acid and furfural which normally released during acid
pretreatment of lignocellulosic biomass were used. Inhibitors have
detrimental effect on enzyme production capability of the fungus
but this novel strain used in present study has minimal effect on
its enzyme production. The analytical results obtained after 120
hours of fermentation were shown in table 1. Results showed that
waste water containing up to 0.1% concentration of all the
inhibitors can be used for enzyme production.
TABLE-US-00001 TABLE NO. 1 Enzyme production in the presence of
inhibitors S. No. Inhibitors Average FPA/ml 1 HMF (0.25%) 6.07 2
Furfural (0.25%) 6.83 3 Acetic acid (0.5%) 6.49 4 Formic acid
(0.1%) 6.87 5 HFAF (0.05%) 6.97 6 HFAF (0.1%) 5.78 7 HFAF (0.2%)
0.014 8 Control 7.43 *HFAF (all the inhibitors in equal
percentage)
Example 3--Utilization of Waste Water for the Production of
Enzymes
[0049] Cultivation and enzyme production from Penicillium
funiculosum MRJ-16 mutant strain was carried out under the
conditions and media components identical in example no. 1 except
that the water used for media preparation was waste water obtained
after hot water washing of rice straw. Water procured after hot
water extraction of rice straw contains sand particles, free
sugars, waxes, phenolics, protein and ash. After 96 hours of
incubation, enzyme broth was harvested and filter paper activity
was analyzed.
TABLE-US-00002 TABLE NO. 2 Utilization of waste water generated
after rice straw pretreatment (Hot water extraction and acid
pre-treatment) for enzyme production Filter paper Hemicellulases/
S. No. Variables activity (FPA/ml) xylanase B-glucosidases 1
Control 7.57 190 60 2 Extractive 7.54 178 55 water
[0050] Production of Enzymes Using Pre-Treated Biomass and Waste
Water Together for Enzyme Production
[0051] Enzyme production from Penicillium funiculosum MRJ-16 strain
was performed using media components and conditions as described in
example no. 1 except the cellulose was replaced with acid
pre-treated rice straw. Pretreatment of rice straw was done at
sulfuric acid concentration from 0.5-1.5% w/w, temperature
110-160.degree. C. for 10-30 min. Slurry obtained after acid
pre-treatment of rice straw containing inhibitors was used as such
after pH rectification for enzyme production. After 120 hours of
cultivation results obtained were 16.2 g/L of protein, 180 IU/ml of
Xylanase/Hemicellulase, 65 IU/ml of .beta.-glucosidase and 6.5
FPU/ml of filter paper activity.
Example 5--Hydrolysis of Pre-Treated Lignocellulosic Biomass
[0052] The efficiency of enzyme produced in example 4 was
determined by its ability to hydrolyze and produce glucose from
lignocellulosic biomass such as acid pretreated rice straw.
Hydrolysis was performed at high substrate loading of acid
pretreated biomass i.e. 15% at pH 4-5, 50 mM citrate buffer,
temperature 50.degree. C. with enzyme loading of 5FPU/g enzyme
loading of dry substrate. Sugars released were determined at
regular interval of time by HPLC. Enzyme cocktail worked
efficiently and leads to 60% glucan conversion in 48 hours.
* * * * *